Differential regulation of anti-inflammatory genes by p38 MAP kinase and MAP kinase kinase 6.

BackgroundConventional p38α inhibitors have limited efficacy in rheumatoid arthritis, possibly because p38 blockade suppresses the counter-regulatory mechanisms that limit inflammation. In contrast, targeting the upstream MAP kinase kinases, MKK3 and MKK6, partially maintains p38-mediated anti-inflammatory responses in bone marrow-derived macrophages (BMDM). In this study, we explored the mechanisms that preserve anti-inflammatory gene expression by evaluating differential regulation of IL-10 and p38-dependent anti-inflammatory genes in MKK3-/-, MKK6-/-, and p38 inhibitor-treated wildtype cells.MethodsBMDM from wild type (WT), MKK3-/-, and MKK6-/- mice were pre-treated with p38 inhibitor SB203580 (SB), JNK inhibitor SP600125 (SP), and/or ERK inhibitor PD98059 (PD) and stimulated with LPS. Supernatant protein levels were measured by multiplex bead immunoassay. mRNA expression was determined by qPCR and protein expression by Western blot analysis. De novo IL-10 mRNA synthesis was quantified in cells treated with ethynyl-uridine and LPS followed by reverse transcription and qPCR. mRNA half-life was measured in LPS-treated cells that were then incubated with actinomycin D ± SB203580.ResultsPre-treatment of WT BMDM with p38 inhibitor significantly reduced IL-10 production in the three groups, while ERK and JNK inhibitors had minimal effects. IL-10 production was significantly decreased in MKK3-/- BMDM compared with either WT or MKK6-/- cells. IL-10 mRNA expression was modestly reduced in MKK3-/- BMDM but was preserved in MKK6-/- cells compared with WT. De novo IL-10 mRNA synthesis was inhibited in MKK3-/- and p38 inhibitor pre-treated cells, but not MKK6-/- cells compared with WT. IL-10 mRNA half-life was markedly reduced in p38 inhibitor-treated WT cells while MKK-deficiency had minimal effect. DUSP1 mRNA levels were preserved in MKK-deficient cells but not in p38 inhibitor-treated WT cells. Tristetraprolin mRNA and protein levels were reduced in p38 inhibitor-treated WT cells compared with MKK6-/- cells.ConclusionUnlike p38-inhibition, the absence of MKK6 mostly preserves IL-10 and TTP protein expression in BMDM. MKK6-deficiency also spares DUSP1 and IL-1RA, which are key negative regulators of the inflammatory response. Together, these data suggest that MKK6 is a potential therapeutic target in RA

Regulation of the JNK pathway by TGF-beta activated kinase 1 in rheumatoid arthritis synoviocytes.

c-Jun N-terminal kinase (JNK) contributes to metalloproteinase (MMP) gene expression and joint destruction in inflammatory arthritis. It is phosphorylated by at least two upstream kinases, the mitogen-activated protein kinase kinases (MEK) MKK4 and MKK7, which are, in turn, phosphorylated by MEK kinases (MEKKs). However, the MEKKs that are most relevant to JNK activation in synoviocytes have not been determined. These studies were designed to assess the hierarchy of upstream MEKKs, MEKK1, MEKK2, MEKK3, and transforming growth factor-beta activated kinase (TAK)1, in rheumatoid arthritis (RA). Using either small interfering RNA (siRNA) knockdown or knockout fibroblast-like synoviocytes (FLSs), MEKK1, MEKK2, or MEKK3 deficiency (either alone or in combination) had no effect on IL-1beta-stimulated phospho-JNK (P-JNK) induction or MMP expression. However, TAK1 deficiency significantly decreased P-JNK, P-MKK4 and P-MKK7 induction compared with scrambled control. TAK1 knockdown did not affect p38 activation. Kinase assays showed that TAK1 siRNA significantly suppressed JNK kinase function. In addition, MKK4 and MKK7 kinase activity were significantly decreased in TAK1 deficient FLSs. Electrophoretic mobility shift assays demonstrated a significant decrease in IL-1beta induced AP-1 activation due to TAK1 knockdown. Quantitative PCR showed that TAK1 deficiency significantly decreased IL-1beta-induced MMP3 gene expression and IL-6 protein expression. These results show that TAK1 is a critical pathway for IL-1beta-induced activation of JNK and JNK-regulated gene expression in FLSs. In contrast to other cell lineages, MEKK1, MEKK2, and MEKK3 did not contribute to JNK phosphorylation in FLSs. The data identify TAK1 as a pivotal upstream kinase and potential therapeutic target to modulate synoviocyte activation in RA

Regulation of peripheral inflammation by spinal p38 MAP kinase in rats.

BackgroundSomatic afferent input to the spinal cord from a peripheral inflammatory site can modulate the peripheral response. However, the intracellular signaling mechanisms in the spinal cord that regulate this linkage have not been defined. Previous studies suggest spinal cord p38 mitogen-activated protein (MAP) kinase and cytokines participate in nociceptive behavior. We therefore determined whether these pathways also regulate peripheral inflammation in rat adjuvant arthritis, which is a model of rheumatoid arthritis.Methods and findingsSelective blockade of spinal cord p38 MAP kinase by administering the p38 inhibitor SB203580 via intrathecal (IT) catheters in rats with adjuvant arthritis markedly suppressed paw swelling, inhibited synovial inflammation, and decreased radiographic evidence of joint destruction. The same dose of SB203580 delivered systemically had no effect, indicating that the effect was mediated by local concentrations in the neural compartment. Evaluation of articular gene expression by quantitative real-time PCR showed that spinal p38 inhibition markedly decreased synovial interleukin-1 and -6 and matrix metalloproteinase (MMP3) gene expression. Activation of p38 required tumor necrosis factor alpha (TNFalpha) in the nervous system because IT etanercept (a TNF inhibitor) given during adjuvant arthritis blocked spinal p38 phosphorylation and reduced clinical signs of adjuvant arthritis.ConclusionsThese data suggest that peripheral inflammation is sensed by the central nervous system (CNS), which subsequently activates stress-induced kinases in the spinal cord via a TNFalpha-dependent mechanism. Intracellular p38 MAP kinase signaling processes this information and profoundly modulates somatic inflammatory responses. Characterization of this mechanism could have clinical and basic research implications by supporting development of new treatments for arthritis and clarifying how the CNS regulates peripheral immune responses

Acid-sensing ion channel 3 decreases phosphorylation of extracellular signal-regulated kinases and induces synoviocyte cell death by increasing intracellular calcium.

IntroductionAcid-sensing ion channel 3 (ASIC3) is expressed in synoviocytes, activated by decreases in pH, and reduces inflammation in animal models of inflammatory arthritis. The purpose of the current study was to characterize potential mechanisms underlying the control of inflammation by ASIC3 in fibroblast-like synoviocytes (FLS).MethodsExperiments were performed in cultured FLS from wild-type (WT) and ASIC3-/- mice, ASIC1-/- mice, and people with rheumatoid arthritis. We assessed the effects of acidic pH with and without interleukin-1β on FLS and the role of ASICs in modulating intracellular calcium [Ca(2+)](i), mitogen activated kinase (MAP kinase) expression, and cell death. [Ca(2+)](i) was assessed by fluorescent calcium imaging, MAP kinases were measured by Western Blots; ASIC, cytokine and protease mRNA expression were measured by quantitative PCR and cell death was measured with a LIVE/DEAD assay.ResultsAcidic pH increased [Ca(2+)](i) and decreased p-ERK expression in WT FLS; these effects were significantly smaller in ASIC3-/- FLS and were prevented by blockade of [Ca(2+)]i. Blockade of protein phosphatase 2A (PP2A) prevented the pH-induced decreases in p-ERK. In WT FLS, IL-1β increases ASIC3 mRNA, and when combined with acidic pH enhances [Ca(2+)](i), p-ERK, IL-6 and metalloprotienase mRNA, and cell death. Inhibitors of [Ca(2+)](i) and ERK prevented cell death induced by pH 6.0 in combination with IL-1β in WT FLS.ConclusionsDecreased pH activates ASIC3 resulting in increased [Ca(2+)](i), and decreased p-ERK. Under inflammatory conditions, acidic pH results in enhanced [Ca(2+)](i) and phosphorylation of extracellular signal-regulated kinase that leads to cell death. Thus, activation of ASIC3 on FLS by acidic pH from an inflamed joint could limit synovial proliferation resulting in reduced accumulation of inflammatory mediators and subsequent joint damage

PUMA-mediated apoptosis in fibroblast-like synoviocytes does not require p53

PUMA (p53-upregulated modulator of apoptosis) is a pro-apoptotic gene that can induce rapid cell death through a p53-dependent mechanism. However, the efficacy of PUMA gene therapy to induce synovial apoptosis in rheumatoid arthritis might have limited efficacy if p53 expression or function is deficient. To evaluate this issue, studies were performed to determine whether p53 is required for PUMA-mediated apoptosis in fibroblast-like synoviocytes (FLS). p53 protein was depleted or inhibited in human FLS by using p53 siRNA or a dominant-negative p53 protein. Wild-type and p53(-/- )murine FLS were also examined to evaluate whether p53 is required. p53-deficient or control FLS were transfected with PUMA cDNA or empty vector. p53 and p21 expression were then determined by Western blot analysis. Apoptosis was assayed by ELISA to measure histone release and caspase-3 activation, or by trypan blue dye exclusion to measure cell viability. Initial studies showed that p53 siRNA decreased p53 expression by more than 98% in human FLS. Loss of p53 increased the growth rate of cells and suppressed p21 expression. However, PUMA still induced apoptosis in control and p53-deficient FLS after PUMA cDNA transfection. Similar results were observed in p53(-/- )murine FLS or in human FLS transfected with a dominant-negative mutant p53 gene. These data suggest that PUMA-induced apoptosis in FLS does not require p53. Therefore, approaches to gene therapy that involve increasing PUMA expression could be an effective inducer of synoviocyte cell death in rheumatoid arthritis regardless of the p53 status in the synovium

Interventions for Infection and Inflammation-Induced Preterm Birth: a Preclinical Systematic Review

Spontaneous preterm births (< 37 weeks gestation) are frequently associated with infection. Current treatment options are limited but new therapeutic interventions are being developed in animal models. In this PROSPERO-registered preclinical systematic review, we aimed to summarise promising interventions for infection/inflammation-induced preterm birth. Following PRISMA guidance, we searched PubMed, EMBASE, and Web of Science using the themes: "animal models", "preterm birth", "inflammation", and "therapeutics". We included original quantitative, peer-reviewed, and controlled studies applying prenatal interventions to prevent infection/inflammation-induced preterm birth in animal models. We employed two risk of bias tools. Of 4020 identified studies, 23 studies (24 interventions) met our inclusion criteria. All studies used mouse models. Preterm birth was most commonly induced by lipopolysaccharide (18 studies) or Escherichia coli (4 studies). Models varied according to infectious agent serotype, dose, and route of delivery. Gestational length was significantly prolonged in 20/24 interventions (83%) and markers of maternal inflammation were reduced in 20/23 interventions (87%). Interventions targeting interleukin-1, interleukin-6, and toll-like receptors show particular therapeutic potential. However, due to the heterogeneity of the methodology of the included studies, meta-analysis was impossible. All studies were assigned an unclear risk of bias using the SYRCLE risk of bias tool. Interventions targeting inflammation demonstrate therapeutic potential for the prevention of preterm birth. However, better standardisation of preterm birth models, including the dose, serotype, timing of administration and pathogenicity of infectious agent, and outcome reporting is urgently required to improve the reproducibility of preclinical studies, allow meaningful comparison of intervention efficacy, and aid clinical translation

Quantitative biomarker analysis of synovial gene expression by real-time PCR

Synovial biomarker analysis in rheumatoid arthritis can be used to evaluate drug effect in clinical trials of novel therapeutic agents. Previous studies of synovial gene expression for these studies have mainly relied on histological methods including immunohistochemistry and in situ hybridization. To increase the reliability of mRNA measurements on small synovial tissue samples, we developed and validated real time quantitative PCR (Q-PCR) methods on biopsy specimens. RNA was isolated from synovial tissue and cDNA was prepared. Cell-based standards were prepared from mitogen-stimulated peripheral blood mononuclear cells. Real time PCR was performed using TaqMan chemistry to quantify gene expression relative to the cell-based standard. Application of the cellular standard curve method markedly reduced intra- and inter-assay variability and corrected amplification efficiency errors compared with the C(t) method. The inter-assay coefficient of variation was less than 25% over time. Q-PCR methods were validated by demonstrating increased expression of IL-1β and IL-6 expression in rheumatoid arthritis synovial samples compared with osteoarthritis synovium. Based on determinations of sampling error and coefficient of variation, twofold differences in gene expression in serial biopsies can be detected by assaying approximately six synovial tissue biopsies from 8 to 10 patients. These data indicate that Q-PCR is a reliable method for determining relative gene expression in small synovial tissue specimens. The technique can potentially be used in serial biopsy studies to provide insights into mechanism of action and therapeutic effect of new anti-inflammatory agents

Elevated autoantibody content in rheumatoid arthritis synovia with lymphoid aggregates and the effect of rituximab

IntroductionThe purpose of this study was to quantitatively evaluate the contribution of synovial lymphoid aggregates to autoantibody (rheumatoid factor [RF] and anti-cyclic citrullinated peptide [anti-CCP]) and total immunoglobulin (IgG and IgM) production in rheumatoid arthritis (RA) patients and the effect thereon of the B-cell-depleting antibody, rituximab, in the ARISE (Assessment of Rituximab's Immunomodulatory Synovial Effects) trial.MethodsAutoantibodies as well as total IgM and IgG were quantified by enzyme-linked immunosorbent assay in extracts of synovial tissues and matched serum from patients with RA or osteoarthritis (OA). Synovial biopsies and serum were obtained at baseline and 8 weeks following rituximab therapy in 14 RA patients. A synovial/serum index (SSI) was calculated as the ratio of synovial to serum antibody/albumin, with values above 1 representing synovial enrichment. Lymphoid aggregates were evaluated histologically.ResultsAnti-CCP IgG, but not RF-IgM, was significantly enriched in RA synovia compared with serum. Total IgM and IgG were also enriched in RA, but not in OA. SSI correlated significantly with mRNA content for both IgM and IgG, demonstrating that it reflected synovial immunoglobulin production. RA synovia with lymphocyte aggregates contained significantly elevated RF-IgM and anti-CCP IgG compared with tissues with diffuse lymphoid infiltration. Rituximab treatment did not affect synovial autoantibody or total immunoglobulin SSI overall. However, in aggregate-containing tissues, rituximab significantly reduced total IgM and IgG SSI as well as IgM and IgG1 mRNA. Surprisingly, RF-IgM and anti-CCP IgG SSIs were unchanged by rituximab in aggregate-containing synovia.ConclusionsCombined with earlier observations that synovial lymphoid aggregates are unaltered by rituximab treatment, these data suggest that lymphoid aggregates may provide a protective niche for autoantibody-producing cells

Capripoxvirus tissue tropism and shedding: A quantitative study in experimentally infected sheep and goats

AbstractSheeppox virus and goatpox virus cause systemic disease in sheep and goats that is often associated with high morbidity and high mortality. To increase understanding of the pathogenesis of these diseases, we undertook quantitative time-course studies in sheep and goats following intradermal inoculation of Nigerian sheeppox virus or Indian goatpox virus in their respective homologous hosts. Viremia, determined by virus isolation and real-time PCR, cleared within 2 to 3 weeks post inoculation. Peak shedding of viral DNA and infectious virus in nasal, conjunctival and oral secretions occurred between 10 and 14 days post inoculation, and persisted at low levels for up to an additional 3 to 6 weeks. Although gross lesions developed in multiple organ systems, highest viral titers were detected in skin and in discrete sites within oronasal tissues and gastrointestinal tract. The temporal distribution of infectious virus and viral DNA in tissues suggests an underlying pathogenesis that is similar to smallpox and monkeypox where greatest viral replication occurs in the skin. Our data demonstrate that capripoxvirus infections in sheep and goats provide additional and convenient models which are suitable not only for evaluation of poxvirus-specific vaccine concepts and therapeutics, but also study of poxvirus–host interactions